Team:Tokyo-NoKoGen/Project/tank

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<h1>Tank</h1>
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<img src="https://static.igem.org/mediawiki/2010/c/c3/Tank_top.png" style="width:300px;">
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<h2>Introduction</h2>
<h2>Introduction</h2>
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“EcoTank” is the shell in a bacterial cell, which is known as bacterial micro compartment (BMC). BMC play a role as reactors in which 1, 2-propandiol or ethanolamine were accumulated and metabolized by the enzyme in the BMC (1, 2, 3) (Fig.1). <br>
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The gene coding enzyme relating to metabolize of these compounds form a operon.  For example, the operon coding to 1,2-propandiol utilization, <i>pdu</i> operon from <i>Citrobacter freundii, </i> is composed of 24 genes, <i>pduA-X</i>. In these genes, <i>pduA, B, J, K, N, U</i> code a shell protein forming BMC. And other genes code enzyme relating to 1, 2-propandiol utilization, some enzymes are located in BMC. (3)
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The other hand, PduP-GFP fusion protein localize in cells having BMC (4). This suggests that fusion protein which has any function such as binding metal was localized in BMC (5) (Fig. 2).<br>
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So, we suggest that BMC can be tank which has function such as accumulating any compounds and isolating them from the cell. This feature enable to capture any target compounds and accumulating it from contamination such as naphthalene from bitumen or metal ion from water or soil.
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<center><img src="https://static.igem.org/mediawiki/2010/0/09/Tank_fig1.png" alt="" style="width:90%;"></center>
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<img src="https://static.igem.org/mediawiki/2010/d/d1/Tank_fig2.png" alt="" style="float:right; width:50%;" />
<h2>Why is this device needed?</h2>
<h2>Why is this device needed?</h2>
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<div align="left">
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EcoTank enables to be ease to purify from other protein in cell. <br>
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Bacterial cell express a lot kinds of protein. But EcoTank enable us to purify objective products from these mixed protein solution. <br clear="right">
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<h2>What is this device composed inside it?</h2>
<h2>What is this device composed inside it?</h2>
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Fig. 3 shows the component of EcoTank.
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BBa K317029 code EcoTank composed of <i>pduA, B, J, K, N, U</i>. <br>
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BBa K317030 and BBa K317031 code chimeric protein of PduP and GFP (PduP-GFP).
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PduP is localized at EcoTank, and labels EcoTank with fluorescent of GFP. (Fig. 2) <br>
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<center><img src="https://static.igem.org/mediawiki/2010/0/0f/Tank_fig3.png" alt="" style="width:95%;" /></center>
<h2>How does this device work in EcoTanker</h2>
<h2>How does this device work in EcoTanker</h2>
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In our EcoTanker project, we cloned the <i>pduA, B, J, K, N, U</i> which code BMC relating to 1, 2,-propandiol utilization, and tried to evaluate the expression of EcoTank as BioBrick.<br>
 +
The tank device helps us to purify the target compounds from contamination.
 +
This device feature is two points. <br>
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<ul>
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<li> 1) The target compounds taken into the cell is accumulated in the tank. </li>
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<li> 2) We can easily purify the tank full of the target compounds by some peptide tag like His tag etc…<br>
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In this study, we construct chimeric protein of PduP which is the enzyme relating to 1, 2,-propandiol utilization and localized in EcoTank and Green fluorescent protein (GFP) in order to evaluate the expression of EcoTank as BioBrick. </li>
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</ul>
<h2>Progress</h2>
<h2>Progress</h2>
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<h3>Construct of new BioBrick</h3>
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<p><img src="https://static.igem.org/mediawiki/2010/2/25/Tank_fig4.jpg" style="float:right; width="15%;" />
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<b>The construction of EcoTank</b>
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Genes of <i>pduA, B, pduJ, K, pduN, pduU </i>were cloned from <i>Citrobacter freundii</i> by PCR. Then the restriction site of <i>Pst</i>Ⅰ on the cloned gene was deleted by PCR. Using the PCR products, BBa K317029 was constructed. This gene was ligated with pSB1A3. <br>
 +
 +
<b>The construction of pduP-GFP</b>
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Using the primer which codes, BBa K317030, BBa K317031 was constructed. The gene was ligated with pSB1C3. <br>
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Then using the plasmid pSB1A3 (BBa K317029) and pSB1C3 (BBa K317030), or pSB1C3 (BBa K317031), BL21(DE3) was transformed (Fig. 4). <br>
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</p>
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<h3>Evaluation</h3>
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<b>Expression of PduP-GFP</b>
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Expression of PduP-GFP was performed by culturing BL21 (DE3) in 100 ml LB broth.
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Then wet cell was prepared from the culture and was homogenized by sonication. Then the soluble fraction was prepared by centrifugation of the homogeneity. Then in order to confirm the expression of PduP-GFP, SDSPAGE was performed. <br>
 +
In the result of SDSPAGE analysis for soluble fraction and insoluble fraction, the band was observed at 29 kDa, this size is PduP-GFP’s one. And the wet cell was green. These results suggest that PduP-GFP show fluorescence in the cell. In this study, we confirm only expression of PduP-GFP as BioBrick<br clear="right"><br>
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BioBricks we submitted are bellows.
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<h3>Perspective</h3>
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In future work, we try to perform co-expression of PduP-GFP and EcoTank in order to confirm the formation of the EcoTank. <br>
 +
The other way, in Fan C. report (2010), the result of PduP-GFP localized in EcoTank suggests that attaching functional protein such as binding metal to protein localized EcoTank enable EcoTank to accumulate any metal. And attaching the functional protein such as his tag or antigen43 to EcoTank enable EcoTank to be ease to be purified..
 +
Moreover, EcoTank can package harmful compounds to cells. This suggests EcoTank is expected to enable to accumulate any compound.
<h1>References</h1>
<h1>References</h1>
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1. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversityand evolution. <i>J Exp Bot</i>. 2003 Feb; 54(383):609-22<br>
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2. C.S. Crowley, M.R. Sawaya, T.A. Bobik and T.O. Yeates, Structure of the PduU shell protein from the Pdu microcompartment of Salmonella, <i>Structure</i> 16 (2008), pp. 1324–1332<br>
 +
3. S. Tanaka, M.R. Sawaya and T.O. Yeates, Structure and mechanisms of a protein-based organelle in Escherichia coli, <i>Science</i> 327 (2010), pp. 81–84.<br>
 +
4. Parsons JB, Dinesh SD, Deery E, Leech HK, Brindley AA, et al. (2008) Biochemical and structural insights into bacterial organelle form and biogenesis. <i>J Biol Chem</i> 283: 14366–14375<br>
 +
5. Fan C, et al.(2010) Short N-terminal sequences package proteins into bacterial microcompartments. Proc Natl Acad Sci USA 107:7509–7514<br>
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Latest revision as of 02:05, 28 October 2010

Introduction

“EcoTank” is the shell in a bacterial cell, which is known as bacterial micro compartment (BMC). BMC play a role as reactors in which 1, 2-propandiol or ethanolamine were accumulated and metabolized by the enzyme in the BMC (1, 2, 3) (Fig.1).
The gene coding enzyme relating to metabolize of these compounds form a operon. For example, the operon coding to 1,2-propandiol utilization, pdu operon from Citrobacter freundii, is composed of 24 genes, pduA-X. In these genes, pduA, B, J, K, N, U code a shell protein forming BMC. And other genes code enzyme relating to 1, 2-propandiol utilization, some enzymes are located in BMC. (3) The other hand, PduP-GFP fusion protein localize in cells having BMC (4). This suggests that fusion protein which has any function such as binding metal was localized in BMC (5) (Fig. 2).
So, we suggest that BMC can be tank which has function such as accumulating any compounds and isolating them from the cell. This feature enable to capture any target compounds and accumulating it from contamination such as naphthalene from bitumen or metal ion from water or soil.

Why is this device needed?

EcoTank enables to be ease to purify from other protein in cell.
Bacterial cell express a lot kinds of protein. But EcoTank enable us to purify objective products from these mixed protein solution.

What is this device composed inside it?

Fig. 3 shows the component of EcoTank. BBa K317029 code EcoTank composed of pduA, B, J, K, N, U.
BBa K317030 and BBa K317031 code chimeric protein of PduP and GFP (PduP-GFP). PduP is localized at EcoTank, and labels EcoTank with fluorescent of GFP. (Fig. 2)

How does this device work in EcoTanker

In our EcoTanker project, we cloned the pduA, B, J, K, N, U which code BMC relating to 1, 2,-propandiol utilization, and tried to evaluate the expression of EcoTank as BioBrick.
The tank device helps us to purify the target compounds from contamination. This device feature is two points.
  • 1) The target compounds taken into the cell is accumulated in the tank.
  • 2) We can easily purify the tank full of the target compounds by some peptide tag like His tag etc…
    In this study, we construct chimeric protein of PduP which is the enzyme relating to 1, 2,-propandiol utilization and localized in EcoTank and Green fluorescent protein (GFP) in order to evaluate the expression of EcoTank as BioBrick.

Progress

Construct of new BioBrick

The construction of EcoTank Genes of pduA, B, pduJ, K, pduN, pduU were cloned from Citrobacter freundii by PCR. Then the restriction site of PstⅠ on the cloned gene was deleted by PCR. Using the PCR products, BBa K317029 was constructed. This gene was ligated with pSB1A3.
The construction of pduP-GFP Using the primer which codes, BBa K317030, BBa K317031 was constructed. The gene was ligated with pSB1C3.
Then using the plasmid pSB1A3 (BBa K317029) and pSB1C3 (BBa K317030), or pSB1C3 (BBa K317031), BL21(DE3) was transformed (Fig. 4).

Evaluation

Expression of PduP-GFP Expression of PduP-GFP was performed by culturing BL21 (DE3) in 100 ml LB broth. Then wet cell was prepared from the culture and was homogenized by sonication. Then the soluble fraction was prepared by centrifugation of the homogeneity. Then in order to confirm the expression of PduP-GFP, SDSPAGE was performed.
In the result of SDSPAGE analysis for soluble fraction and insoluble fraction, the band was observed at 29 kDa, this size is PduP-GFP’s one. And the wet cell was green. These results suggest that PduP-GFP show fluorescence in the cell. In this study, we confirm only expression of PduP-GFP as BioBrick

Perspective

In future work, we try to perform co-expression of PduP-GFP and EcoTank in order to confirm the formation of the EcoTank.
The other way, in Fan C. report (2010), the result of PduP-GFP localized in EcoTank suggests that attaching functional protein such as binding metal to protein localized EcoTank enable EcoTank to accumulate any metal. And attaching the functional protein such as his tag or antigen43 to EcoTank enable EcoTank to be ease to be purified.. Moreover, EcoTank can package harmful compounds to cells. This suggests EcoTank is expected to enable to accumulate any compound.

References

1. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversityand evolution. J Exp Bot. 2003 Feb; 54(383):609-22
2. C.S. Crowley, M.R. Sawaya, T.A. Bobik and T.O. Yeates, Structure of the PduU shell protein from the Pdu microcompartment of Salmonella, Structure 16 (2008), pp. 1324–1332
3. S. Tanaka, M.R. Sawaya and T.O. Yeates, Structure and mechanisms of a protein-based organelle in Escherichia coli, Science 327 (2010), pp. 81–84.
4. Parsons JB, Dinesh SD, Deery E, Leech HK, Brindley AA, et al. (2008) Biochemical and structural insights into bacterial organelle form and biogenesis. J Biol Chem 283: 14366–14375
5. Fan C, et al.(2010) Short N-terminal sequences package proteins into bacterial microcompartments. Proc Natl Acad Sci USA 107:7509–7514

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